plants Article Genome Wide Identification and Comparative Analysis of the Serpin Gene Family in Brachypodium and Barley Shazia Rehman 1,2,3,* , Bodil Jørgensen 3, Ejaz Aziz 4, Riffat Batool 5, Samar Naseer 6 and Søren K. Rasmussen 3,* 1 Department of Botany, Rawalpindi Women University, 6th Road, Satellite Town, Rawalpindi 46200, Pakistan 2 Department of Botany, Govt. Gordon College Rawalpindi, Rawalpindi 46000, Pakistan 3 Department of Plant and Environmental Sciences, Faculty of Sciences, University of Copenhagen, 1871 Frederiksberg C, Denmark; [email protected] 4 Department of Botany, Government Degree College Khanpur, Haripur 22650, Pakistan; [email protected] 5 University Institute of Biochemistry and Biotechnology, PMAS, Arid Agriculture University, Rawalpindi, Rawalpindi 46300, Pakistan; riff[email protected] 6 Department of Biology and Environmental Science, Faculty of Sciences, Allama Iqbal Open University, Islamabad 44000, Pakistan; [email protected] * Correspondence: [email protected] (S.R.); [email protected] (S.K.R.) Received: 3 October 2020; Accepted: 15 October 2020; Published: 26 October 2020 Abstract: Serpins (serine protease inhibitors) constitute one of the largest and most widely distributed superfamilies of protease inhibitors and have been identified in nearly all organisms. To gain significant insights, a comprehensive in silico analysis of the serpin gene family was carried out in the model plant for temperate grasses Brachypodium distachyon and barley Hordeum vulgare using bioinformatic tools at the genome level for the first time. We identified a total of 27 BdSRPs and 25 HvSRP genes in Brachypodium and barley, respectively, showing an unexpectedly high gene number in these model plants. Gene structure, conserved motifs and phylogenetic comparisons of serpin genes supported the role of duplication events in the expansion and evolution of serpin gene family. Further, purifying selection pressure was found to be a main driving force in the evolution of serpin genes. Genome synteny analysis indicated that BdSRP genes were present in syntenic regions of barley, rice, sorghum and maize, suggesting that they evolved before the divergence of these species from common ancestor. The distinct expression pattern in specific tissues further suggested a specialization of functions during development and in plant defense. These results suggest that the LR serpins (serpins with Leu-Arg residues at P2–P10) identified here can be utilized as candidates for exploitation in disease resistance, pest control and preventing stress-induced cell death. Additionally, serpins were identified that could lead to further research aimed at validating and functionally characterizing the role of potential serpin genes from other plants. Keywords: serpin proteins; phylogenetic comparison; gene duplication; selection pressure; genome synteny 1. Introduction The serpin superfamily is a member of the most ubiquitous and successful class of inhibitors and is found in all organisms, including animals, plants, bacteria, viruses and Archaea [1–3]. The majority of serpins inhibit serine proteases of the chymotrypsin family by employing a unique “suicide substrate” mechanism of irreversible inhibition [4,5], but few of them have evolved an ability to inhibit cysteine Plants 2020, 9, 1439 ; doi:10.3390/plants9111439 www.mdpi.com/journal/plants Plants 2020, 9, 1439 2 of 19 proteases as well [1,6]. Some other serpins have bifunctional activity or no inhibitory activity [7]. In general, serpins are large proteins (340–440 aa) with a molecular weight of 40–45 KDa, and they contain a flexible bait loop that can form covalent and irreversible complexes with proteases [1]. A typical inhibitory serpin consists of several α-helices and β-strands, together with an external reactive center loop (RCL). The RCL region is usually composed of 20–24 amino acid residues and is of critical importance in determining the inhibitory capacity of serpins [8]. Structural studies revealed that the P1–P10 sessile bond in the RCL acts as bait for attacking proteases and the inhibitory specificity of serpins is mainly dependent on the identity of the active site residues (P4–P40), in particularly P1 [1,4,9]. Thus, high diversity in these RCL residues may allow the serpins to target a wide range of proteases with different proteolytic specificities. Several fundamental biological processes, including blood coagulating pathway and many other important proteolytic cascades in mammals, are controlled by serpins [10]. Serpin genes characterized so far from plants have inhibitory properties similar to that of animals, but target proteases for plant serpins have not been identified. In plants, serpins play an important role in defense against plant pathogens, having a great potential as breeding targets to improve disease resistance, as well as in food security, since they also appear vital to grain development. For instance, serpins identified in Arabidopsis, soybean and maize have been investigated for activity against the necrotrophic fungus Botrytis cinerea [11]. Similar defensive functions have been suggested against insects and pathogens for the serpins found in high concentrations in cereal grains and apple seeds [12–14]. In cereals, biotic stress-responsive serpins are likely to play an important role in disease resistance against Fusarium culmorum, which causes Fusarium head blight disease in barley [15]; Magnaporthe oryzae, which causes rice blast [16] and Puccinia striiformis, which causes stripe rust in wheat [17]. From pumpkin (Cucurbita maxima) phloem, Yoo et al. [18] isolated the CmPS-1 serpin, which has elastase-inhibiting activity, and they showed a negative correlation between increased levels of CmPS-1 and survival of the green peach aphid. Consistent with these results, an Arabidopsis serpin 1 (AtSerpin1) was shown to significantly reduce the growth of Spodoptera littoralis larvae and may be a good candidate for pest control [19]. A similar study has been carried out for three sorghum serpins (Sbser1, Sbser2 and Sbser3) showing activity against corn earworm [20]. These results suggest that plant serpins may inhibit the digestive protease activity of insects and the proteolytic enzymes of pathogens. Based on previous studies on cereals, serpins are considered essential for grain development and quality. In barley, serpins act as storage proteins during grain filling and contribute up to 5% to the 7% lysine content of the total grain [1,21], and hence were suggested to be a target for breeding high-lysine barley. Wheat and rye grain serpins have evolved to inhibit proteases specifically adapted to the breakdown of grain prolamins [12]. Furthermore, serpins from barley and wheat grains have been assessed for inhibitory activity against chymotrypsin and cathepsin G. Thus, serpins found in grains participate in grain development by providing protection to storage proteins from digestion by insects and fungi [12,13]. Plant serpins are also found to participate in plant immunity as negative regulators of stress-induced cell death under biotic and abiotic stresses [22]. For example, AtSerpin1 was shown to act on Metacaspase 9 (AtMC9) in vitro and in vivo with the cysteine protease RD21 and to regulate stress-induced cell death in response to fungal attack [23–25]. More recently, AtSRP4 and AtSRP5 were identified as negative regulators of stress-induced cell death caused by bacteria [26]. Two other serpins, AtSRP2 and AtSRP3, were found to be associated with the regulation of growth responses in the presence of alkylating agents [27]. In another example, Bhattacharjee et al. [28] demonstrated that downregulation of the OsSRP-LR serpin (rice serpin1) shows exaggerated cell death upon exposure to pathogen infection, UV light and saline conditions. In line with this observation, Dhanushkodi et al. [29] reported increased papain-like cysteine protease activity, early nodule senescence and reduced plant growth with knockdown of the LR-type serpin (MtSer6) gene in Medicago truncatula under drought-stressed conditions. Hence, it was suggested that serpins constitute an important line of defense in plants under biotic and abiotic stresses. Plants 2020, 9, 1439 3 of 19 The majority of plant serpins identified until now are known to be inhibitory and rarely perform non-inhibitory functions. In animals, the non-inhibitory serpins have diverse functions, including roles as hormone transporters [30], molecular chaperones [31] or tumor suppressors [32]. The presence of non-inhibitory serpins in plants may indicate their possible role in hormone regulation (as hormone transport molecules), protein storage or protein folding (as chaperones) [9]. In a recent study, Cohen and Fluhr [33] described the non-inhibitory function of a barley serpin Z4 for the first time and reported increased β-amylase activity due to interaction with serpin Z4 in response to heat and oxidative stresses. Both serpin Z4 and β-amylase are abundant seed proteins in many barley cultivars [34]. Thus, serpin Z4 also exhibits chaperone-like activity as well as an inhibitory function, demonstrating the dual biological role for cereal serpins [35,36]. A technical aspect of serpins is that protein Z is the dominant protein in beer foam, probably contributing to the foam stability and barley malt varieties with a high protein Z content can be selected for with the use of molecular markers [37]. Although serpin genes have been characterized from many plant species [38,39], a genome-wide comparison of the serpin gene